Malaria is the scourge of many developing countries, particularly those in sub-Saharan Africa, claiming several million lives each year. Malaria is caused by mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium. Four species of Plasmodium protozoa (P. falciparum, P. vivax, P. ovale and P. malariae) are responsible for the disease in humans; many others cause disease in animals, such as P. yoelii and P. berghei. P. falciparum accounts for the majority of lethal infections in humans. Researchers have struggled for decades to make a successful subunit or attenuated whole-organism vaccine but with limited success. Factors that have hampered the development of a subunit vaccine include the complexity of the malaria life cycle, the wide variety of immune response induced by the malaria parasite, and an incomplete knowledge of protective immunity. In contrast, attenuated whole-organism vaccines are better understood and in principle should provide full protective immunity.
Upon introduction into the bloodstream by the female Anopheline mosquito during blood feeding, the infectious sporozoite of Plasmodium invades and multiplies within the hepatocyte. Recognition and invasion of a hepatocyte is a complex process involving traversal through macrophage-like Kupffer cells (1) and several hepatocytes (2) before forming a parasitophorous vacuole (PV) in the final hepatocyte. Currently, only a few proteins of sporozoites have been described that play an essential role in establishing infection of the liver but are thought to be conserved in all species of Plasmodium. These include circumsporozoite protein (CS), thrombospondin-related anonymous protein (TRAP), microneme proteins essential for cell traversal (SPECT1, SPECT2 or PPLP1, and CelTOS) and PbIMC1a, which variously are involved in motility of sporozoites, recognition of surface receptors on host cells, and traversal and invasion of host cells (3-10). Within the PV, the sporozoite transforms and grows (trophozoite stage) and multiplies (schizont stage) for a period of a few days, resulting in the generation and release of thousands of merozoites that invade red blood cells.
The current rationale for the characterization of Plasmodium molecules involved in liver infection lies in the development of a (subunit) vaccine that protects against liver and subsequent blood stage infection (11). Immunization studies using complete radiation-attenuated sporozoites (RAS) showed full protection against subsequent challenge with infectious sporozoites in both animal models of malaria and in human volunteers (12). The protective immunity that is observed after immunization with RAS requires that the sporozoites infect hepatocytes and transform into the trophozoite stage (13). Such immunity is mediated by complex mechanisms involving antibody responses that inhibit sporozoite motility and host cell invasion and T cell responses directed against intrahepatocytic stages. CD4+ T helper cells and cytotoxic CD8+ T lymphocytes recognizing MHC presented parasite-derived peptides, as well as cytokines (IL-2, IFN-γ, and IL-12; TNF-α, IL-1, and IL-6) and nitric oxide, have been shown to be critical effectors in protection against preerythrocytic malaria (13-15).
However, vaccination studies with subunit vaccines that contain only (parts of) single proteins of sporozoites have failed so far to provide any significant, long-lasting protective immunity (11, 12). The lack of significant progress with subunit vaccines stimulated recent attempts to produce a vaccine based on the nonreplicating, metabolically active RAS (12). However, such vaccines suffer from significant drawbacks, not least the question of safety and reproducibility because the amount of radiation that generates the attenuated state is strictly defined. Parasites that are underirradiated remain infectious, and those that are overirradiated do not induce protective immunity. Recently, it has been shown that genetically attenuated sporozoites (GAS) that lack sporozoite-specific conserved genes (uis3 and uis4) that are apparently important for sporozoite development in the hepatocyte can induce significant or complete protective immunity in the P. berghei rodent model of malaria when different immunization protocols are used (16, 17). The use of GAS for vaccination might remove the uncertainty associated with RAS once a more thorough understanding of the mechanisms of immunity invoked by GAS and their developmental defect(s) are available.
Although the currently developed uis3− and uis4− Plasmodium mutants show promising results, there is always need for better and/or alternative genetically attenuated mutants that give a protective response in malaria.